Saccharides (also known as sugars or carbohydrates), amino acids, fatty acids and nucleotides comprise the more major building blocks of biological macromolecules such as oligosaccharides, proteins, lipids and nucleic acids, respectively. Due to the diversity within a group of building blocks, such as saccharides for example, and the variety of ways in which to order the building blocks of a group or groups, large numbers of structurally distinct biological macromolecules are possible. Oligosaccharides alone, for example, are a group of biological polymers which comprise an extremely diverse group of molecules. Oligosaccharides exist as individual compounds as well as components of larger compounds. For example, a combination of an oligosaccharide and a protein is termed a glycoprotein. Similarly, a combination of an oligosaccharide and a lipid is termed a glycolipid. Oligosaccharides, glycoproteins and glycolipids have a large number of functions in nature. For example, these macromolecules in general, and their saccharide components in specific, serve as recognition molecules in a wide variety of normal and abnormal biological processes, including cancer and inflammation.
Because of the critical importance of oligosaccharides, alone or in combination with other molecules, there has been great interest in determining the structures of oligosaccharides and in making new oligosaccharides as well as portions of known oligosaccharides. An oligosaccharide is composed of individual saccharides, also known as monosaccharides. Typically, monosaccharides possess five carbon atoms (pentoses), six carbon atoms (hexoses), or are variants thereof. Regardless of whether there are five or six carbon atoms, each monosaccharide is capable of existing in five-atom ring forms (also known as five-membered rings or furanoses) and six-atom ring forms (also known as six-membered rings or pyranoses). For example, shown below are the five-membered ring (structure on left side) and the six-membered ring (structure on right side) for glucose: ##STR1## In addition, in solution, monosaccharides are in equilibrium between ringed forms (in which there is an oxygen in the ring) and open chain forms in which there is an aldehyde ("aldehydo") group in place of the bond between carbon-1 ("C-1") and the former ring oxygen. For example, shown below are ringed and open chain forms for glucose: ##STR2## Depending upon the arrangement of the substituents at C-1, the ring form may be the .alpha. anomer or .beta. anomer as shown above. Since aldehydo groups may be reduced (i.e., converted to a lower oxidation state such as an alcohol), a monosaccharide which is capable of existing in an open chain aldehydo form is considered a reducing monosaccharide.
In all oligosaccharides, two or more individual saccharides (i.e., monosaccharides) are linked together to form an oligosaccharide. In oligosaccharides which bear a reducing monosaccharide, the other monosaccharides are always linked together with the linkage from C-1 of one monosaccharide to one of C-2, C-3, C-4, C-5 or C-6 of another monosaccharide. For example, shown below is an oligosaccharide in which glucose ("Glc") is linked to glucose from C-1 of the glucose on the left side to C-4 of the glucose on the right side: ##STR3## The linkage between the two glucose molecules may be .alpha. or .beta., depending upon the arrangement of the substituents at C-1. The glucose on the right side of the di-glucose oligosaccharide structure depicted above possesses an OH group (at C-1) which may exist as an aldehydo group and, therefore, is termed the "reducing end" of the oligosaccharide. Conversely, the glucose on the left side of the di-glucose structure depicted above does not possess an OH at C-1 and, therefore, is termed the "non-reducing end" of the oligosaccharide. In oligosaccharides that are linear (i.e., monosaccharides linked in a straight chain without branching), there will be one reducing end and one non-reducing end. However, if there is branching in an oligosaccharide (i.e., more than one monosaccharide is linked to a given monosaccharide), there will still be only one reducing end but two or more non-reducing ends. Since each monosaccharide may be linked to different positions of the given monosaccharide, there is the potential for oligosaccharides of significant complexity.
The current method for structural determination of oligosaccharides is based upon removal of monosaccharides from the non-reducing end(s). This approach is hampered by the fact that an analysis based upon removal from the non-reducing end(s) is a subtractive approach (i.e., compares the total monosaccharides, and the nature of their linkages, before and after removal to determine what is missing) and by the fact that where an oligosaccharide possesses more than one non-reducing end, additional information is necessary before the locations of the multiple non-reducing end monosaccharides may be affixed. Similarly, because a method for sequential removal of monosaccharides from the reducing end of an oligosaccharide has not been available, the preparation of new oligosaccharides or the isolation of portions of pre-existing oligosaccharides after removal of one or more monosaccharides from the reducing end of pre-existing oligosaccharides has not been possible. Thus, there is a need in the art for methods which permit the sequential removal of monosaccharides from the reducing end of oligosaccharides. The present invention fulfills these needs and further provides other related advantages.